Central Dogma Lecture 15 RNA processing, splicing, and degradation

Question 1

What processing of pre-mRNA needs to be done in bacteria?

Answer 1

RNAs are generally “ready to go”

Question 2

How are RNAs transcribed in eukaryotes?

Answer 2

As precursors that need to be processed to yield final useful RNA

Question 3

How do mRNAs need to be processed in humans?

Answer 3

-Capping
-Splicing
-Polyadenylation
-Export to cytoplasm
-Other RNAs are edited and bases are modified

Question 4

What is the 5’ cap on mRNA

Answer 4

-A modification of the 1st mRNA nucleotide with 7-methylguanosine
-Added co-transcriptionally to mRNA

Question 5

What is the purpose of the 5’ cap in mRNA?

Answer 5

-Protects the mRNA from nucleases
-Binds to specific complexes of proteins
-Recruits the ribosome for translation

Question 6

How is the 5’ cap added?

Answer 6

-5’ cap is a 7-methylguanosine
-Initially the 5’ end has a triphosphate (pppNpNp…)
-Phosphohydrolase removes γ phosphate (ppNpNp…)
-Guanylytransferase uses GTP tp add a G (GpppNpNp…)
-Guanine-7-methyltransferase adds a methyl (m^7GpppNpNp)
-2’-O-methyltransferase adds another methyl (m^7GpppmNpNp…)

Question 7

Where is the 5’ cap on mRNA

Answer 7

-7-methylguanosine is added “backwards” via an unusual 5’,5’-triphosphate linkage
-First two bases are also often methylated
-Not methylated in yeast
-Methylated in human cells

Question 8

What is an intron?

Answer 8

-Named for intervening sequences
-Removed from mRNA

Question 9

What is an “exon”?

Answer 9

-Named for expressed sequences
-Kept in mRNA

Question 10

Introns in yeast

Answer 10

Only a minority of genes have introns

Question 11

Introns in humans characteristics

Answer 11

-Most genes have introns, often genes average 8 introns/gene
-Introns are ~10x longer than exons in humans
-Exons usually <200 bp, introns can be 50-20,000 bp
-Genes can be 100,000 bases long and take hours to transcribe
-Average human gene is 8,t00 bp, 90% introns

Question 12

What is the purpose of introns?

Answer 12

-Not yet known
-Probably not junk
-Possibly alternative splicing of genes provides diversity
-Allow for rapid protein evolution through domain addition/subtraction

Question 13

Introns allow for alternative splicing to make isoforms

Answer 13

-Muscle protein α-tropomyosin gene has 7 isoforms
-Exon usage depends on presence of splicing factors in each tissue

Question 14

Four classes of introns

Answer 14

-Group I
-Group II
-Eukaryotic mRNA introns
-tRNA introns

Question 15

Group I and Group II introns

Answer 15

-Self-splicing
-First example of RNA catalysis
-Require no additional proteins or ATP
-In nuclear, mitochondrial, chloroplast, and phage genomes

Question 16

Eukaryotic mRNA introns

Answer 16

-Spliced by catalytic RNAs (snRNPs) in spliceosomes
-The most common introns in humans
-Active site resembles Group II ribozyme
-Lots of ATP-dependent conformational switches

Question 17

tRNA introns

Answer 17

-Spliced by protein enzymes
-Primary transcript cleaved by endonuclease
-Exons are joined by ATP-dependent ligase

Question 18

What organism was used when RNA self-splicing was discovered?

Answer 18

-Used Tetrahymena thermophila
-Eukaryote easy to grow, has 20k copies of a particular gene that gets spliced

Question 19

How was RNA self-splicing discovered

Answer 19

-mRNA to be spliced, MgCl2 and GTP, and nuclear extract (because they assumed they needed an enzyme) were added to a tube
-In gel electrophoresis for the negative control, they left out the nuclear extract and there was just as much splicing
-Eventually found that RNA can act as an enzyme, “ribozyme”

Question 20

Self-splicing mechanism of group I introns

Answer 20

-Cell will splice two sequences together
-RNA folds into a shape that bods exogenous GTP (GMP or G also works)
-3’-OH of GTP attacks 5’ splice site
-GTP leaves and G at 3’ end of protein slips into a pocket
-Free 3’OH of the exon attacks the downstream splice site
-Releases the intron (modified with G) plus the ligated exons

Question 21

Self-splicing of Group II introns

Answer 21

-Similar to group I introns, except:
-Nucleophile in the first step is the 2’-OH of internal A (not 3’ OH because A is aimed at the 5’ splice site by secondary structure, can’t just attack anywhere)
-Formation of a “lariat loop structure, in which the A has three phosphodiester bonds and one is a 2’,5’-phosphodiester linkage

Question 22

How’s does the overview of spliceosomal intron splicing compare to Group II?

Answer 22

-Similar to Group II splicing
-Uses 2’-OH of an internal A within the intron as a nucleophile
-Forms a lariat intermediate
-The 3’-OH of newly revealed 3’ end attacks splice site
-Differs from Group II because exogenous proteins and RNAs are required
-Complex is called the spliceosome

Question 23

What is the spliceosome and what is it made up of?

Answer 23

-A large complex that helps with mRNA splicing
-Made up of multiple specialized RNP complexes called small nuclear ribonucleoproteins (snRNPs) and dozens of other proteins

Question 24

What are snRNAs?

Answer 24

-100-200 nucleotides long and make up snRNPs
-U1, U2, U4, U5, and U6 are abundant in the nuclei

Question 25

How is the spliceosome formed?

Answer 25

-snRNPs contain snRNAs with sequences complementary to pre-mRNA
-U1 helps define the 5’ splice site
-U2 binds the branch site, near the 3’ end of the intron
-Results in A bulging to be the nucleophile
-A forms 2’,5’- phosphodiester bond of the lariat-like intermediate
-Then U4, U5, U6, and 80 other proteins form spliceosome
-ATP is required for assembly and conformational switching but not chemistry of cleavage
-Some parts are attached to the CDT (carboxy-terminal domain) of RNAP II, coordinating splicing with transcription

Question 26

How are group II self-splicing introns and the spliceosome related?

Answer 26

-Chemical events of splicing are identical in mechanism
-Spliceosome may have evolved from Group II self-splicing introns
-Spliceosome may have eveolved to help with huge introns

Question 27

What is polyadenylation?

Answer 27

Addition of the poly(A) tail

Question 28

What is the poly(A) tail?

Answer 28

-String of A residues added to the 3’ end of most eukaryotic mRNAs
-~30 residues in yeast and 50-100 in animals
-Serves as binding site for specific proteins
-May help protect mRNA from enzymatic destruction

Question 29

How are poly(A) tails added?

Answer 29

-RNA Pol II synthesizes RNA beyond the region encoding cleavage signal sequences
-One element that is highly conserved is “AAUAAA”
-This cleavage signal sequence is bound by an enzyme complex that includes an endonuclease and polyadenylate polymerase
-All of these are tethered to the CTD in RNA Pol II
-Endonuclease cleaves RNA 10-30nt downstream of highly conserved AAUAAA, leaving 3’-OH
-Polyadenylate polymerase synthesizes 80-250 nt of A using ATP as substrate
-It’s not RNA Pol II terminating that determines the end of the transcript

Question 30

How can a single gene yield different RNA products?

Answer 30

-Transcripts can be alternatively spliced
-Occurs often in humans, could account for increasing “complexity” in eukaryotes
-Cleavage/polyadenylation patterns can vary, yielding different mature transcripts
-In diverse eukaryotes, RNA can be “edited” (bases removed/added)

Question 31

Alternative splicing, calcitonin

Answer 31

-Calcitonin and calcitonin-gene-related peptide, in rat thyroid and brain, respectively, made from same mRNA
-Primary transcript has two poly(A) sites; one predominates in the brain, other in thyroid
-In brain, splicing eliminates the calcitonin exon (exon 4); in the thyroid, this exon is retained
-Resulting peptides are processed further to yield the two proteins in respective cells

Question 32

What are some characteristics of rRNAs and tRNAs?

Answer 32

-Not capped or polyadenylated
-Cleaved (slicing) from longer precursors
-Have bases that are modified in post-transcriptional reactions

Question 33

What bases are modified in post-transcriptional reactions in tRNAs and rRNAs?

Answer 33

-Meythylation (base or 2’-ribose)
-Pseudouridine (𝜓, uridine is removed, rotated, put back)
Thiouridine (gets a sulfur)

Question 34

Pre-rRNA processing in bacteria

Answer 34

-Similar in bacteria and humans
-Several enzyme-mediated cleavages and modification

Question 35

Post-transcriptional processing of tRNA

Answer 35

-tRNAs are derived from longer RNA precursors and extensively modified
-5’ and 3’ ends are cleaved, bases modified, and “CCA” is added
-CCA is added to the 3’ end by CCA-adding enzyme; binds the amino acid
-Yeast tRNA^Tyr gets bottom anticodon segment spliced out

Question 36

How long do cellular mRNAs last?

Answer 36

-RNA lifetime is another means of gene regulation
-Half-lives vary from seconds to hours
-Typical vertebrate mRNA ~3 hrs (10 turnovers per cell generation)
-Shorter (~1.5 min) Half-lives for bacterial mRNAs

Question 37

mRNAs and degradation

Answer 37

-In eukaryotes, mRNAs can be degraded via deadenylation (removal of poly-A tail)
-When poly is less than or equal to 10 nucleotides, mRNAs are subject to de-capping
-Decapped and deadenylated RNAs are degraded by RNases: Xrn1 (5′→3′)/exosome (3′→5′)